Stabilized 1,25-dihydroxyvitamin d2 and method of making same

ABSTRACT

A stabilized 1,25-dihydroxyvitamin D 2  composition which is particularly well suited for pharmaceutical formulations, pharmaceutical formulations of the 1,25-dihydroxyvitamin D 2  composition, and a method of making the purified composition by purifying a crude 1,25-dihydroxyvitamin D 2  from acetone/water, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

The benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication Ser. No. 61/036,021 filed on Mar. 12, 2008 is herebyclaimed.

BACKGROUND

1. Field of the Disclosure

The disclosure relates generally to active vitamin D hormones inpurified form. More particularly, the disclosure relates to stabilized1,25-dihydroxyvitamin D₂ which is particularly well suited forpharmaceutical formulations.

2. Brief Description of Related Technology

Vitamin D compounds having a hydroxy group at the 1-α position, oftenconsidered to be activated vitamin D compounds, have had considerableattention in recent years because of their potent activities. Theseactivated vitamin D compounds are, however, also known as beingchemically unstable, particularly under exposure to light and to oxygen,and as having poor storage stability at higher temperatures. Thecompounding of activated vitamin D compounds into pharmaceuticalformulations can exacerbate these stability problems.

U.S. Pat. No. 6,903,083 (Jun. 7, 2005) reported purifying24-hydroxyvitamin D₂ and cis-1α-hydroxyvitamin D₂ by recrystallizationfrom methyl formate, and results of stability testing thereof.

SUMMARY

One aspect of the disclosure provides a surprisingly stable1,25-dihydroxyvitamin D₂, characterized by being free of methyl formate,preferably completely free of methyl formate.

Another aspect of the disclosure provides an efficient method forcrystallizing 1,25-dihydroxyvitamin D₂ to produce a stabilized1,25-dihydroxyvitamin D₂ at relatively high yield, the method includingcrystallization from an acetone/water mixture.

For the compositions and methods described herein, preferred features,such as components, compositional ranges thereof, functional properties,substituents, conditions, and steps, can be selected from the variousexamples provided herein.

Further aspects and advantages will be apparent to those of ordinaryskill in the art from a review of the following detailed description,taken in conjunction with the drawings. While the composition and methodare susceptible of embodiments in various forms, the descriptionhereafter includes specific embodiments with the understanding that thedisclosure is illustrative, and is not intended to limit the inventionto the specific embodiments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

For further facilitating the understanding of the present invention, twodrawing figures are appended hereto.

FIGS. 1 and 2 depict accelerated stability testing of stabilized1,25-dihydroxyvitamin D₂ according to the disclosure herein compared to1,25-dihydroxyvitamin D₂ containing methyl formate.

DETAILED DESCRIPTION

The stabilized 1,25-dihydroxyvitamin D₂ is characterized by being freeof methyl formate and having a high level of purity (e.g. at least 97%).The method for preparing the stabilized 1,25-dihydroxyvitamin D₂ caninclude crystallization from an acetone/water mixture.

The stabilized 1,25-dihydroxyvitamin D₂ is and method of crystallizationare contemplated to encompass embodiments including any combination ofone or more of the additional optional elements, features and stepsfurther described below (including those shown in the figures) unlessstated otherwise.

The stabilized 1,25-dihydroxyvitamin D₂ is crystalline, storage stableand well suited for modern therapy formulations. Studies havedemonstrated that stabilized 1,25-dihydroxyvitamin D₂ according to thedescription herein is surprisingly stable. In particular, it ischaracterized by a reduced rate of degradation on heat challenge andduring prolonged storage. The stabilized 1,25-dihydroxyvitamin D₂according to the description herein will exhibit excellent,unprecedented storage stability when formulated in a pharmaceuticaldosage form, e.g., a soft gelatin capsule.

The stabilized 1,25-dihydroxyvitamin D₂ has a purity equal to or greaterthan 97% (i.e., at least 97%) based on total area under the curve (AUC)by high performance liquid chromatography (HPLC), preferably at least98% purity or at least 98.5% purity. It is also free of methyl formate.The stabilized 1,25-dihydroxyvitamin D₂ substance optionally has a lowconcentration of 1,25-dihydroxyprevitamin D₂ (e.g. preferably not morethan 2.5%, not more than 1%, or not more than 0.5%).

Methyl formate is taught in the prior art as a suitable crystallizationsolvent for 1,25-dihydroxyvitamin D₂ and many other active vitamin Dcompounds. It has now been found that residual methyl formate has adeleterious effect on stability of 1,25-dihydroxyvitamin D₂ and itsprevitamin, leading to marked degradation over time, even at very lowlevels of residual methyl formate concentration and under controlledstorage conditions (e.g. −20° C. and inert atmosphere). Withoutintending to be bound by any particular theory, it is believed thatmethyl formate hydrolyzes (e.g., at elevated temperature, or over time),in the presence of even a very small amount of water, to produce formicacid and methanol. Further without intending to be bound by anyparticular theory, it is believed that formic acid promotes theformation of 1,25-dihydroxyvitamin D₂ by-products presumably by causingthe formation of a conjugated diene via protonation of the 25-hydroxygroup and subsequent removal of water.

Accordingly, in another aspect the stabilized 1,25-dihydroxyvitamin D₂will optionally be further free of or substantially free of residualsolvents which can produce acids including, but not limited to, alcoholesters of organic acids and alcohol esters of inorganic esters.Nonlimiting examples include formate esters (e.g., methyl formate, ethylformate); acetate esters (e.g., methyl acetate, butyl acetate, ethylacetate, isoamyl acetate, 1,2-ethanediol diacetate, 1,2,3-propanetrioltriacetate) and lactate esters (e.g., ethyl lactate, methyl lactate).For example, the level of such residual components is preferably lessthan 1%, 0.5%, 1000 ppm, 500 ppm, or 100 ppm. The stabilized1,25-dihydroxyvitamin D₂ is also optionally free of or substantiallyfree of one or more of the following: halogenated solvents,dimethylsulfoxide, dimethylformamide, and acids. For example, the levelof such residual halogenated solvents is preferably less than 1%, 0.5%,1000 ppm, 500 ppm, or 100 ppm.

Further optionally, but preferably, the stabilized 1,25-dihydroxyvitaminD₂ according to the description herein has a purity of at least 97% (orat least 98% or at least 98.5%), a residual solvents content of 0.5% orless, further optionally a total impurities content of 1.5% or less, andfurther optionally has no single impurity greater than 0.5% by HPLC.

The stabilized 1,25-dihydroxyvitamin D₂ according to the descriptionherein is stable under conditions of heat challenge. In one embodiment,the 1,25-dihydroxyvitamin D₂ is at least about 98%, preferably at least98.5%, for example at least 99% pure after storage at 60° C. for atleast 24 hours, preferably for at least 72 hours. In another embodiment,the amount of 1,25-dihydroxyvitamin D₂ that remains after storage at 60°C. for at least 24 hours preferably is at least 97.5%, more preferablyat least 99%, for example at least 99.9% of the initial amount. Inanother embodiment, the amount of 1,25-dihydroxyvitamin D₂ that remainsafter storage at 60° C. for at least 72 hours preferably is at least97%, more preferably at least 98.5%, for example at least about 99.5% ofthe initial amount.

The stabilized 1,25-dihydroxyvitamin D₂ according to the descriptionherein is stable over a prolonged period of time at long term storageconditions. In one embodiment, the stabilized 1,25-dihydroxyvitamin D₂according to the description herein has a purity of at least 98%,preferably at least 98.5%, more preferably at least 99% after storage at−20° C. under argon for at least 3, 6, 9, 12, or 15 months. Embodimentsand examples of the percentage of the initial amount of1,25-dihydroxyvitamin D₂ that remains after storage at −20° C. underargon for at least 3, 6, 9, 12, or 15 months are shown in Table 1.

TABLE 1 Percentage of the initial amount of 1,25-dihydroxyvitamin D₂that remains after a prolonged period of time Period of Time PreferablyMore preferably Example At least 3 months At least 99% At least 99.5% Atleast 99.9% At least 6 months At least 97% At least 98.5% At least 99.9%At least 9 months At least 94% At least 97% At least 99.9% At least 12months At least 94% At least 97% At least 99.9% At least 15 months Atleast 94% At least 97% At least 99.9%

The stabilized 1,25-dihydroxyvitamin D₂ is suitably used inpharmaceutical formulations, such as for oral use (e.g., soft or hardgelatin capsules, solutions, tablets) and for parenteral use. Thus, thepharmaceutical formulation made from or containing stabilized1,25-dihydroxyvitamin D₂ can be in the form of a non-aqueous solution ora non-aqueous suspension (e.g. contained in a vial or ampoule) or insolid form. In one preferred embodiment, the stabilized1,25-dihydroxyvitamin D₂ formulation is in an oil (e.g. fractionatedcoconut oil). In another preferred embodiment, the stabilized1,25-dihydroxyvitamin D₂ formulation is a solid in the form of a tablet,a capsule, a granule, or a powder. The pharmaceutical formulation cancomprise, consist essentially of, or consist of stabilized1,25-dihydroxyvitamin D₂ together with one or more pharmaceuticallyacceptable excipients. Pharmaceutically acceptable excipients includefunctional and non-functional ingredients including carriers, diluents,processing aids, and release-modifying agents. Such pharmaceuticalformulations can be made with the stabilized 1,25-dihydroxyvitamin D₂substance described herein, using any suitable technique including knowntechniques.

The dosage of 1,25-dihydroxyvitamin D₂ for oral or parenteraladministration generally is about 0.1 μg per week to 100 μg per week,preferably about 0.7 μg per week to about 70 μg per week, which can besplit into daily or other periodic doses, such as three times per weekfor administration concomitant with hemodialysis. In exemplaryembodiments, a parenteral dosage equivalent to about 0.5 μg per day toabout 7 μg per day is contemplated, while an oral dosage equivalent toabout 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg per day iscontemplated.

The stabilized 1,25-dihydroxyvitamin D₂ can be dispensed by unit dosageform comprising about 0.1 μg to about 25 μg in a pharmaceuticallyacceptable carrier per unit dosage, for example about 1 μg to about 10μg or about 1 μg to about 5 μg. A sustained-release or delayed,sustained-release unit dosage form, including about 1 μg to about 10 μg,or more preferred about 3 μg to about 5 μg, for example, is alsocontemplated.

For example, in a soft gelatin formulation, the capsule fill suitablycontains stabilized 1,25-dihydroxyvitamin D₂ which preferably isdissolved in a pharmaceutically acceptable oil, e.g., fractionatedcoconut oil, and includes an antioxidant which may be, for example,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) orvitamin E. The capsule shell preferably contains gelatin, glycerin,titanium dioxide and coloring agent. The fill is typically about 30-90%by weight of the whole capsule, preferably about 40-70% by weight.

The 1,25-dihydroxyvitamin D₂ compound may be prepared by any of theknown methods of synthesis. Crude 1,25-dihydroxyvitamin D₂ can be columnpurified to remove the trans isomer according to known methods. Acolumn-purified 1,25-dihydroxyvitamin D₂ can be stabilized byrecrystallization from a suitable organic solvent or mixture of organicsolvent and water consistent with the disclosure herein, e.g., acetoneand water. The recrystallized crystals are then preferably ground to afine powder and dried in a vacuum oven (e.g. at 40° C. for 8-12 hours)to remove residual solvents. One or more successive (generally at leasttwo) recrystallizations can be performed. Suitable solvents, in additionto acetone/water, include acetonitrile, butanol, diethyl ketone,ethanol, ether, ethylene glycol, heptane, methanol, methyl ethyl ketone,propanol, tetrahydrofuran, water, and combinations thereof. Ifrecrystallizations are performed from acid-containing or acid-yieldingsolvents such as methyl formate, then one or more successive (generallyat least two) recrystallizations will be performed with a preferredsolvent according to the disclosure herein, to remove undesired residualcomponents.

Partially pure 1,25-dihydroxyvitamin D₂ preferably is crystallized froma solvent that is free of methyl formate, preferably further free of atleast one of the following solvents selected from the group consistingof formate ester solvents, acetate ester solvents, lactate estersolvents, alcohol ester solvents, halogenated solvents,dimethylsulfoxide, dimethylformamide, and solvents capable of generatingacid. The solvent system is preferably acetone/water, more preferablyusing a 1:1 ratio. While other solvents may be used, it was found thatcrystallization from acetone/water gave a very high yield of desiredproduct and also beneficially provided very low levels of1,25-dihydroxyprevitamin D₂. Each acetone/water crystallization step wasfound to give greater than 90% yield compared to methyl formatecrystallization which was found to give yields in the range of 50% to60%.

If a multi-stage crystallization method is performed to purify the1,25-dihydroxyvitamin D₂, then preferably the preferred solventsidentified herein (e.g., free of methyl formate, preferably further freeof at least one of the following solvents selected from the groupconsisting of formate ester solvents, acetate ester solvents, lactateester solvents, alcohol ester solvents, halogenated solvents,dimethylsulfoxide, dimethylformamide, and solvents capable of generatingacid) are used in the final crystallization stage, with the goal ofminimizing and preferably at least substantially eliminating non-desiredspecies identified herein, such as residual solvents.

The crude material is first refluxed with acetone at a temperature in arange of 50° C. to 60° C. (15 ml/1 g) until a clear solution isobtained. The solution is then filtered through a sintered funnel and avolume of water (preferably an equal volume) is gradually added. Oncethe temperature is reduced to about 25° C. degrees and crystallizationbegins, the mixture is further chilled (e.g. flask placed at 4° C.freezer for 24 h). The solid is then filtered and washed, preferablywith an acetone/water mixture (e.g. 1:1 ratio). Preferably, theacetone/water mixture is pre-chilled, e.g. to 4° C. The crystallizationcan be repeated until the product meets the desired purityspecifications, preferably to a level of at least 97% purity, morepreferably at least 98% or at least 98.5% purity by HPLC.

The HPLC analysis can suitably follow the parameters in Table 2 below.

TABLE 2 Column: YMC-Pack Pro C18, 5 μm, 12 nm, 250 × 4.6 mm Mobilephase: 70:30 acetonitrile: water isocratic Flow rate: 1.5 ml per minuteWave length: 265 nm Run time: 20 min Sample temp: 15° C. ± 5° C. Columntemp: 30° C. ± 5° C. Sample concentration: 1 mg/ml Injection volume: 10μL

EXAMPLES

The following examples are provided for illustration and are notintended to limit the scope of the invention.

Example 1 Preparation of Stabilized 1,25-Dihydroxyvitamin D₂

1,25-dihydroxyvitamin D₂ is prepared following the procedure describedin the Journal of Organic Chemistry 1988, 53, 3450-3457. The crudematerial is treated with maleic anhydride in tetrahydrofuran at roomtemperature, followed by column purification, to remove the trans isomervia a Diels Alder adduct.

Partially pure 1,25-dihydroxyvitamin D₂ was crystallized fromacetone/water (1:1). The crude material was first refluxed with HPLCgrade acetone (15 ml/1 g) until a clear solution was obtained. It wasthen filtered and an equal volume of 18.2 megaohm water was graduallyadded. Once the temperature came down to about 25° C. degrees andcrystal formation started, the flask was placed in a 4° C. freezer for24 h. The solid was filtered and washed with pre-chilled 1:1acetone/water at 4° C. The crystallization was repeated until theproduct met the desired purity specifications. In this example, thepurity was 99.4% (HPLC); single known impurity not more than 0.5%(1β,25-dihydroxyvitamin D₂, 5,6-trans-1,25-dihydroxyvitamin D₂,22-cis-1,25-dihydroxyvitamin D₂, and 1,25-dihydroxyprevitamin D₂);single unknown impurity not more than 0.1%. To dry, the solid materialwas ground via a mortar and pestle and placed at 40° C. under vacuum for8-12 hours.

The parameters in Table 1 above were used for the HPLC analysis.

Example 2 Accelerated Stability Testing of Stabilized1,25-Dihydroxyvitamin D₂

Samples of stabilized 1,25-dihydroxyvitamin D₂ according to Example 1and commercially-obtained 1,25-dihydroxyvitamin D₂ (SAFC, Madison, Wis.USA) made by crystallization from methyl formate were placed in a 60° C.oven in several 2 ml amber vials with Teflon-lined caps. Samples weretaken out at different time points and purity was checked by HPLCaccording to Example 1. The results are presented in Tables 3 and 4below and in FIGS. 1 and 2. FIG. 1 shows the observed stability of thetwo preparations over the 24 hours, and FIG. 2 shows stability over 72hours. From these studies it is clear that the purified materialprepared according to Example 1 was surprisingly more stable compared tothe commercially available material prepared using methyl formate andcontaining residual methyl formate. The stabilized material preparedaccording to the Example 1 will clearly have advantage when any heat isrequired during formulation.

TABLE 3 methyl formate At 60° C. 0 h 4 h 8 h 16 h 24 h 48 h 72 hCommercial 500 ppm Purity 96.80 95.18 94.98 94.69 93.98 93.86 93.82Example 1 not detected (absolute %) 99.40 99.38 99.42 99.33 99.42 99.3198.89

TABLE 4 methyl formate At 60° C. Initial % 4 h 8 h 16 h 24 h 48 h 72 hCommercial 500 ppm Purity 100 98.33 98.12 97.82 97.09 96.97 96.92Example 1 not detected (% of initial 100 99.98 100 99.93 100 99.90 99.50amount)

Example 3 Long-Term Stability Testing of Stabilized1,25-Dihydroxyvitamin D₂

Additional samples of 1,25-dihydroxyvitamin D₂ (SAFC, Madison, Wis. USA)that were substantially free of 1,25-dihydroxyprevitamin D₂ were made bycrystallization from methyl formate and having varying levels ofresidual methyl formate. These samples were stored at −20° C. underargon and periodically tested for purity by HPLC. Other samples of1,25-dihydroxyvitamin D₂ made according to the disclosure herein andwithout methyl formate were also made and tested under the sameconditions. The results are shown in Tables 5 and 6 below.

TABLE 5 Batch Purity of 1,25-Dihydroxyvitamin D₂ (absolute %)1,25-dihydroxyvitamin 0 3 6 9 12 15 D₂ months months months monthsmonths months A 10 ppm methyl formate, 99.48% 97.99% 0.11% 1,25- (3½ M)dihydroxyprevitamin D₂ B 16 ppm methyl formate,  99.8% 93.76% no 1,25-(11 M) dihydroxyprevitamin D₂ C 78 ppm methyl formate, 97.54% 94.3%0.04% 1,25- (7 M) dihydroxyprevitamin D₂ D 4061 ppm methyl 95.55% 94.36%80.98% formate, 0.21% 1,25- dihydroxyprevitamin D₂ E no methyl formate, 97.7% 97.7% no 1,25- dihydroxyprevitamin F no methyl formate,  99.5% 99.5%  98.7% 2% 1,25- dihydroxyprevitamin D₂ G no methyl formate,99.36% 99.42% 99.40% no 1,25- dihydroxyprevitamin H no methyl formate,99.40% 99.4% 99.39% no 1,25- dihydroxyprevitamin

TABLE 6 Purity of 1,25-Dihydroxyvitamin D₂ Batch (% of the initialamount) 1,25-dihydroxyvitamin 3 6 9 12 15 D₂ Initial % months monthsmonths months months A 10 ppm methyl formate, 100% 98.50% 0.11% 1,25-(3½ M) dihydroxyprevitamin D₂ B 16 ppm methyl formate, 100% 93.95% no1,25- (11 M) dihydroxyprevitamin D₂ C 78 ppm methyl formate, 100%96.68%   0.04% 1,25- (7 M) dihydroxyprevitamin D₂ D 4061 ppm methyl 100%98.75% 84.75% formate, 0.21% 1,25- dihydroxyprevitamin D₂ E no methylformate, 100% 100% no 1,25- dihydroxyprevitamin F no methyl formate,100%   100%  99.2% 2% 1,25- dihydroxyprevitamin D₂ G no methyl formate,100%   100% 100% no 1,25- dihydroxyprevitamin H no methyl formate, 100%100%   100% no 1,25- dihydroxyprevitamin

Besides the exceptions noted at 3½ months, 7 months, and 11 months, thesamples were tested at zero, 3 months, 6 months, 9 months, 12 months,and 15 months after manufacture. Quite surprisingly, samples exceeding99% purity at the time of manufacture, and even having low amounts of1,25-dihydroxyprevitamin D₂ (e.g., 0.11% or 0%) but containing even asmall amount of residual methyl formate (e.g., 10 ppm or 16 ppm) werefound to fall below 98% purity within 3½ months and below 94% purity by11 months. In contrast, 1,25-dihydroxyvitamin D₂ prepared according tothe invention crystallized from acetone/water and containing no methylformate (batches G & H described above) showed absolutely no decrease inpurity over at least fifteen months of storage.

The foregoing description is given for clearness of understanding only,and no unnecessary limitations should be understood therefrom, asmodifications within the scope of the invention may be apparent to thosehaving ordinary skill in the art.

Throughout the specification, where compositions are described asincluding components or materials, it is contemplated that thecompositions can also consist essentially of, or consist of, anycombination of the recited components or materials, unless describedotherwise.

The practice of a method disclosed herein, and individual steps thereof,can be performed manually and/or with the aid of electronic equipment.Although processes have been described with reference to particularembodiments, a person of ordinary skill in the art will readilyappreciate that other ways of performing the acts associated with themethods may be used. For example, the order of various of the steps maybe changed without departing from the scope or spirit of the method,unless described otherwise. In addition, some of the individual stepscan be combined, omitted, or further subdivided into additional steps.

All patents, publications and references cited herein are hereby fullyincorporated by reference. In case of conflict between the presentdisclosure and incorporated patents, publications and references, thepresent disclosure should control.

1. A stabilized 1,25-dihydroxyvitamin D₂ composition, characterized by apurity of at least 97% based on total AUC by an HPLC assay and beingfree of methyl formate.
 2. (canceled)
 3. The stabilized1,25-dihydroxyvitamin D₂ composition according to claim 1, wherein theamount comprising 2.5% by weight or less of 1,25-dihydroxyprevitamin D₂based on the total weight of the 1,25-dihydroxyvitamin D₂.
 4. (canceled)5. The stabilized 1,25-dihydroxyvitamin D₂ composition according toclaim 1, wherein the amount of total residual solvent is 1.0% or less.6. The stabilized 1,25-dihydroxyvitamin D₂ composition according toclaim 1, being free of acids and substantially free of at least onesolvent selected from the group consisting of formate ester solvents,acetate ester solvents, lactate ester solvents, alcohol ester solvents,halogenated solvents, dimethylsulfoxide, dimethylformamide, and solventscapable of generating acid at elevated temperature or over time. 7.(canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled) 12.The stabilized 1,25-dihydroxyvitamin D₂ composition according to claim1, wherein the purity is at least 98% after the composition is stored atabout 60° C. for a period of at least 24 hours.
 13. The stabilized1,25-dihydroxyvitamin D₂ composition according to claim 1, wherein atleast about 97.5% of the initial amount of the 1,25-dihydroxyvitamin D₂remains after the composition is stored at about 60° C. for a period ofat least 24 hours.
 14. The stabilized 1,25-dihydroxyvitamin D₂composition according to claim 1, wherein the purity is at least about98% after the composition is stored at about −20° C. under argon for aperiod of at least 6 months.
 15. The stabilized 1,25-dihydroxyvitamin D₂composition according to claim 1, wherein at least about 97% of theinitial amount of the 1,25-dihydroxyvitamin D₂ remains after thecomposition is stored at about −20° C. under argon for a period of atleast 6 months.
 16. (canceled)
 17. (canceled)
 18. The stabilized1,25-dihydroxyvitamin D₂ composition according to claim 1, wherein thepurity is at least about 98% after the composition is stored at about−20° C. under argon for a period of at least 12 months.
 19. (canceled)20. A pharmaceutical composition comprising an active component which isthe stabilized 1,25-dihydroxyvitamin D₂ composition of claim 1 and apharmaceutically acceptable excipient.
 21. The composition of claim 20,which is selected from the group consisting of a tablet, a soft gelatincapsule, a non-aqueous solution, and a non-aqueous suspension. 22.(canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)
 26. Thecomposition of claim 20, in unit dosage form, having a content of activecomponent of 0.1 μg to 25 μg.
 27. The composition of claim 21,comprising a solution of an effective amount of the stabilized1,25-dihydroxyvitamin D₂ in fractionated coconut oil, the solutioncontained in a soft gelatin capsule and further comprising anantioxidant selected from the group consisting of butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin E, andcombinations thereof.
 28. (canceled)
 29. (canceled)
 30. (canceled) 31.(canceled)
 32. (canceled)
 33. (canceled)
 34. The composition of claim20, comprising a solid pharmaceutical preparation of an effective amountof the stabilized 1,25-dihydroxyvitamin D₂ composition, wherein thesolid pharmaceutical preparation is in the form of a tablet, a capsule,a granule or a powder.
 35. (canceled)
 36. (canceled)
 37. (canceled) 38.(canceled)
 39. A method for purifying crude 1,25-dihydroxyvitamin D₂,comprising crystallizing 1,25-dihydroxyvitamin D₂ from a solvent systemfree of methyl formate.
 40. The method of claim 39, wherein the solventsystem is further free of at least one of the following solventsselected from the group consisting of formate ester solvents, acetateester solvents, lactate ester solvents, alcohol ester solvents,halogenated solvents, dimethylsulfoxide, dimethylformamide, and solventscapable of generating acid.
 41. The method of claim 40, wherein thesolvent system is free of formate ester solvents, acetate estersolvents, lactate ester solvents, alcohol ester solvents, halogenatedsolvents, dimethylsulfoxide, dimethylformamide, and solvents capable ofgenerating acid.
 42. The method of claim 39, comprising performing saidcrystallization as the final crystallization stage in a multi-stagecrystallization purification.
 43. The method of claim 39, wherein thesolvent system is an acetone/water mixture.
 44. (canceled) 45.(canceled)
 46. (canceled)
 47. The method according to claim 43,comprising refluxing crude 1,25-dihydroxyvitamin D₂ with HPLC gradeacetone; filtering off solids; adding a volume of 18.2 megaohm water tothe acetone solution; and reducing the temperature of the solution tocrystallize 1,25-dihydroxyvitamin D₂.
 48. The method according to claim43, further comprising washing the crystallized 1,25-dihydroxyvitamin D₂with an acetone/water solution and drying the crystallized1,25-dihydroxyvitamin D₂ to remove residual solvents.
 49. (canceled) 50.(canceled)